Method of culturing cells to produce a tissue sheet

ABSTRACT

The disclosure provides methods and systems for tissue engineering including an apparatus and methods for the growth, maintenance, and use of robust tissue sheets using tissue manipulation devices. The tissue manipulation devices provide a method of anchoring the tissue sheets to the cell culture substrate to promote prolonged maturation and subsequent increased mechanical strength. The tissue manipulation devices also provide a technique to facilitate removal of the sheet from the culture container and subsequent production steps to assemble more complex three dimensional organs from the robust sheet. The tissue manipulation devices also facilitate automated handling of the sheets in the assembly processes. The disclosure provides methods and composition derived from robust human sheets to build tissue engineered blood vessels, heart valves, stents, and endarterectomy patches and the like. The use of these tissue manipulation devices facilitates the assembly of complex tissue engineered organs using the novel sheet-based tissue engineering approach.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119 toprovisional application Ser. Nos. 60/355,283, filed Feb. 7, 2002, and60/340,964, filed Dec. 11, 2001, the disclosures of which areincorporated herein by reference. The present application is alsorelated to International Application Serial No. PCT/US02/39789, filedDec. 11, 2002, entitled, “TISSUE ENGINEERED CELLULAR SHEETS, METHODS OFMAKING AND USE THEREOF,” the disclosure of which is incorporated herein.

TECHNICAL FIELD

This invention relates to tissue engineering, and more particularly itrelates to an apparatus and methods for the growth, maintenance, and useof tissue sheets with a tissue manipulation device.

BACKGROUND

There are currently efforts to construct tissue grafts in vitro toovercome the many problems posed by currently used artificialextra-corporeal and implanted devices. Such tissue engineeringtechniques include the creation, design, and fabrication of biologicalprosthetic devices, in combination with synthetic or natural materials,for the augmentation or replacement of body tissues and organs. However,the use of synthetic materials often results in the release of productsand by-products in vivo that induce inflammation, lead to the productionof inflammatory mediators, and may induce autoimmune disorders. The useof natural materials such as bovine collagen and decellularizedextracellular matrix material from xenogeneic and allogeneic sourcespose a risk of passing on pathogens to a recipient, including, suchpathogens obtained from both human materials (e.g., HIV and HBV) andnon-human materials (e.g., prions associated with bovine materials, andthe like). In short, the failure mechanism of most tissue-engineeredorgans is associated directly with the presence of synthetic materials,which trigger various foreign body responses. This is particularly trueof tissue engineered vascular grafts that must operate in the mostimmune sensitive environment in the body.

Vascular disease is typically associated with a severe narrowing ofcoronary and/or peripheral arteries which compromise organ function byrestricting the flow of blood to downstream organs. There are threetreatment strategies to repair these diseased arteries. The simplestrepair is a catheter-based therapy called angioplasty, where aninflatable balloon is introduced via catheter to the damaged area andthen expanded, thus disrupting the atherosclerotic plaque. Althoughthese procedures are relatively inexpensive and pose little threat tothe patient, angioplasty is associated with very poor long-term patencyrates. A more effective version of this treatment involves the placementof a plastic deformable metallic stent inside the artery. This stent,when expanded, helps to hold the artery open after the balloon isremoved. The primary limitation associated with stenting is that thesynthetic material (usually nickel based steel, stainless steel, orNitenol) used for the stent initiates a chronic inflammatory responseand triggers a migration of cells toward the lumen of the blood vessel.This process, called intimal hyperplasia, results in a second narrowingof the diseased artery (restenosis). The third treatment option is asurgically placed bypass graft, which re-routes blood flow around theblockage through a new conduit, ideally made from a vein or arteryharvested from another site in the subject's own body. In large diametervessels (≧6 mm inside diameter) these bypass conduits can also be madefrom synthetic materials such as ePTFE. In most cases, clinicaltreatment strategies try the relatively non-invasive catheter basedangioplasty/stenting before advancing to bypass surgery.

Although bypass procedures are known to have the highest long-termefficacy, the cost and risk associated with such an invasive surgicalprocedure often dictates that stenting is attempted as the primarytreatment method.

In an attempt to increase the efficacy of stenting, new-generationstents have been developed that are coated with drug/protein-impregnatedpolymers. As the polymer resorbs, the drug, which discourages local cellmigration or proliferation (intimal hyperplasia), is eluted into theblood stream. These stents have demonstrated phenomenal success rates inmid-term clinical studies (0–2 years), but, their long-term efficacyafter the protein coating is completely resorbed is in question. Morerecently, advances in cell biology and genetic modifications have givenrise to another generation of stent technologies, called cell seeded orliving stents. In this configuration, cells are seeded onto the strutsof the stent and then implanted. Although few clinical studies have beenpublished on this technology, three limitations exist. First, relativelyfew cells can be loaded onto the small struts of the stent. Second, whenthe stent is expanded, the struts slide relative to each other, thusscraping many of the cells from the stent. Third, the cell coating doesnot offer any sort of membrane to either reduce the inflammatoryresponse to the foreign material or to provide a barrier to prevent cellmigration through the stent to the lumen of the vessel.

SUMMARY

The invention provides a tissue culture method. The tissue culturemethod comprising culturing a population of adherent cells in a tissueculture container in the presence of at least one tissue manipulationdevice, under conditions that allow the formation of a tissue sheetcomprised of living cells and extracellular matrix formed by the cells,whereby the tissue sheet is in contact with the at least one tissuemanipulation device; and removing the tissue sheet from the culturecontainer.

The invention also provides a system for manipulation of a tissue invitro. The system comprises a culture container comprising at least onewall, a bottom, and an upper opening, each of the at least one wall andthe bottom having an inner and outer surface thereby forming an insideand an outside, the at least one wall and bottom comprised of abiocompatible material; at least one tissue manipulation devicecomprising a first end and a second end and at least one wall, whereinthe at least one tissue manipulation device is substantially elongatedfrom the first end to the second end, the at least one tissuemanipulation device in juxtaposition with the at least one wall and/orbottom of the culture container; growing a tissue comprising apopulation of adherent cells in the culture container in the presence ofthe at least one tissue manipulation device such that the population ofadherent cells grow to form a tissue sheet in contact with at least onetissue manipulation device and wherein the cells contact the inside ofat least one wall of the vessel; and removing the tissue from the vesselusing the at least one tissue manipulation device.

The invention provides a genetically engineered living stent. Thegenetically engineered living stent is generated by a method comprisingculturing a population of adherent cells in a culture container in thepresence of at least one tissue manipulation device under conditions toallow the formation of a tissue sheet comprised of cells andextracellular matrix formed by the cells in contact with the at leastone tissue manipulation device whereby the at least one tissuemanipulation device anchors the tissue sheet in the culture containerand wherein at least one cell of the population of cells is transfectedwith an exogenous polynucleotide such that the exogenous polynucleotideexpresses a product. The tissue sheet is removed from the culturecontainer and formed into a tubular structure thereby forming agenetically engineered living stent.

The invention also provides a method of forming a genetically engineeredliving stent. The method includes culturing a population of adherentcells in a culture container in the presence of at least one tissuemanipulation device under conditions to allow the formation of a tissuesheet comprised of cells and extracellular matrix formed by the cells incontact with the at least one tissue manipulation device whereby the atleast one tissue manipulation device anchors the tissue sheet in theculture container and wherein at least one cell of the population ofcells is transfected with an exogenous polynucleotide such that theexogenous polynucleotide expresses a product. The method furtherincludes removing the tissue sheet from the culture container and formedinto a tubular structure thereby forming a genetically engineered livingstent.

The invention further provides a genetically engineered tissue sheetprepared in vitro. The genetically engineered tissue sheet comprises apopulation of adherent cells wherein at least one cell of the populationof cells is transfected with an exogenous polynucleotide such that theexogenous polynucleotide expresses a product and culturing thepopulation of adherent cells in a culture container in the presence ofat least one tissue manipulation device such that the population ofadherent cells grow to form a tissue sheet in contact with the at leastone tissue manipulation device whereby the tissue sheet is anchoredwithin the culture container by the at least one tissue manipulationdevice.

Also provided by the invention is a method for manipulation of agenetically engineered tissue sheet prepared in vitro. The methodcomprises culturing a population of adherent cells in a culturecontainer in the presence of at least one tissue manipulation deviceunder conditions to allow the formation of a tissue sheet comprised ofcells and extracellular matrix formed by the cells in contact with theat least one tissue manipulation device whereby the at least one tissuemanipulation device anchors the tissue sheet in the culture containerand wherein at least one cell of the population of cells is transfectedwith an exogenous polynucleotide such that the exogenous polynucleotideexpresses a product; removing the tissue sheet from the culturecontainer; and forming a desired tissue structure.

The invention provides a living stent generated by a method comprisingculturing a population of adherent cells in a culture container in thepresence of at least one tissue manipulation device under conditions toallow the formation of a tissue sheet comprised of cells andextracellular matrix formed by the cells in contact with the at leastone tissue manipulation device whereby the at least one tissuemanipulation device anchors the tissue sheet in the culture container;removing the tissue sheet from the culture container; and contacting abiocompatible tubular device with the tissue sheet to form one or morelayers of the tissue sheet in contact with the tubular structure therebyforming a living stent.

The invention further provides a composition, comprising a biocompatiblestent comprising a therapeutic agent and a tissue sheet cultured invitro substantially enveloping the biocompatible stent, wherein thetissue sheet is derived from a population of adherent cells cultured ina culture container in the presence of at least one tissue manipulationdevice, wherein the population of adherent cells grow to form the tissuesheet whereby the tissue sheet is in contact with the at least onetissue manipulation device.

The invention also provides a composition, comprising a tissue sheet ofcells formed into a tubular structure by rolling the tissue sheet uponitself one or more times. The tissue sheet is derived from a populationof adherent cells cultured in a culture container in the presence of atleast one tissue manipulation device, wherein the population of adherentcells grow to form the tissue sheet anchored by the at least one tissuemanipulation device.

The invention yet further provides a living stent. The living stent isgenerated by a method comprising culturing a population of adherentcells in a culture container under conditions to allow the formation ofa tissue sheet comprised of living cells and extracellular matrix formedby the cells; removing the tissue sheet from the culture container; andforming a tubular structure with the tissue sheet by wrapping the tissuesheet one or more times around a tubular structure which is mounted on atemporary support mandrel, thereby forming a living stent.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1( a) is a schematic of a sample of tissue produced by sheet-basedtissue engineering utilizing tissue control rods and methods accordingto the invention. The control rods (10) shown by arrows around theperiphery of the tissue (20) are integrated into the tissue and allowthe tissue to be held and easily manipulated. Typically, the sheet isapproximately 10–200 microns thick.

FIG. 1( b) is a photograph of a tissue sheet supported by two tissuecontrol rods.

FIG. 2 is a tissue sheet-wrapped stent supported by a clamping system ina maturation bioreactor.

FIG. 3 is a tissue sheet supported by a ring control rod in a ciruclar6-well plate.

DETAILED DESCRIPTION

As one beneficial aspect of the invention there is provided techniquesto culture a subject's own cells into a robust tissue sheet. Theinvention provides compositions and methods to generate sheets ofautologous and/or allogenic cells. These tissue sheets are robust andcan be shaped or molded into a variety of organs, including bloodvessels, heart valves, and the like. The cells in the sheet can bemanipulated into a desired shape or structure prior to or duringimplantation to assist in the replacement of organs or to assist in thereconstruction and/or healing of an existing organ. Examples of tissuesapplicable to the techniques of the invention include vascular tissue,skin tissue, hepatic tissue, pancreatic tissue, neuronal tissue,urogenital tissue, gastrointestinal tissue, and musculoskeletal tissue.In addition, the cells in a robust tissue sheet can be geneticallymodified to express a diagnostic and/or a therapeutic product (e.g.,polypeptides or polynucleotides) to generate a genetically engineeredtissue graft.

One use of the techniques and compositions of the invention include, forexample, the treatment of cardiovascular diseases and disorders. Currenttreatment strategies for blocked coronary arteries typically includedifferent percutaneous interventions such as balloon angioplasty orstenting, or surgical bypass of the blocked artery. Balloon angioplastyis a catheter-based therapy where flow is restored through the blockedartery by inflating a balloon in the area afflicted with anatherosclerotic lesion. By breaking up the blockage, the vessel isreopened and blood flow is restored. In stenting, the balloonangioplasty is followed by the placement of an expandable metallic stentthat holds the artery open after the balloon is removed. The advantageto these procedures is that they are catheter based, meaning they areless invasive and do not require open-heart surgery. In most cases,physicians will opt for one of the less invasive percutaneous (orcatheter-based) approaches first. The downside to these procedures isthat success rates beyond 6 months are as low as 50%. Most short-termfailures are associated with thrombosis (the formation of blood clots onthe inside of the vessel) due to the lack of a functional endothelialcell lining in the stent. Most long-term failures are caused by cellmigration and in growth into the lumen of the vessel in a process calledintimal hyperplasia.

More recently, however, advances in stenting technology have improvedshort- to mid-term patency rates. By adding controlled release drugcoatings to the stents, cell in-growth that causes secondary closure(restenosis) of the vessel is diminished. Specifically, these stents areloaded with therapeutic agents such as Paclitaxel, rapamycin, heparin,or sirolimus to reduce local cell migration and proliferation. Earlyresults with these drug eluting stents demonstrates lower restenosiswithin 6–9 months. However, the long-term efficacy with thesedrug-eluting stents is still questionable. As the drug degrades, it isquite possible that restenosis rates will increase. Another limitationto the stents is that the body recognizes the stent and polymer coatingas a foreign body and initiates a foreign body inflammatory/immuneresponse. This inflammatory/immune response contributes to long-termfailures by triggering cell migration and subsequent intimalhyperplasia.

The invention provides methods and compositions that can be used totreat vascular diseases and disorders. In one aspect a cellular tissuesheet of the invention is rolled into a cylindrical or tubular structureto provide a very robust cylindrical tissue similar to a blood vessel orother organ comprising a lumen. Cell sheets of the invention comprisedonly of human fibroblasts can form cylindrical grafts with ademonstrated burst pressure greater than about 4000 mmHg (30 timesphysiological blood pressure). This cylindrical tissue sheet can be usedas a stent, thus eliminating the need for a metallic stent. Moreover, itis possible to use the tissue sheet-based construct to perform apercutaneous bypass. In this embodiment, the construct is delivered viacatheter by piercing through the wall of the healthy vessel near theproximal end of a diseased area of the vessel and bypassing the diseasedarea by re-entering the vessel on the distal end of the diseased area. Apercutaneous bypass would not be possible with current technologies,since normal stents do not have a membrane that would allow blood topass through without leaking.

Although described herein with particular reference to formation ofvascular blood vessels, it should be understood that the tubularstructures, generated by rolling a tissue sheet of the invention one ormore times to generate a structure comprising a lumen surrounded by asheet of cells, can be used to form lumens for passage of a variety offluids, not just blood. Other fluids include, but are not limited to,urine, bile, and lymph.

A tissue is a multicellular organization of cells. Bodily tissuecomprises a variety of cell types but typically have a population ofcells of a defined lineage (sometimes referred to as parenchymal cells).In vitro tissues can be generated by culturing a population of cellsunder conditions that allow the cells to multiply and expand undertissue culture conditions. The cells present in a tissue culture may bethe same or may comprise a combination of various cell types including,for example, stromal cells (e.g., fibroblasts or mesenchymal precursorcells) alone or in combination with various parenchymal cells of abodily tissue (e.g., smooth muscle cells, endothelial cells,hepatocytes, keratinocytes, and the like). A tissue sheet comprises apopulation of cells having the same or different morphologies and/orlineages cultured in vitro under conditions whereby the cells grow andmultiply.

The robust tissue sheets are special constructs. They are different fromother standard cell cultures in many ways both structurally andphysically. Structurally: 1) tissue sheet are comprised of multiplelayer of cells, 2) the cells are embedded in a large amount ofextracellular matrix proteins produced by the cells themselves, 3) theextracellular matrix proteins are “natural” in as much as they are notphysically/chemically modified by extraction/isolation, procedures, 4)the extracellular matrix proteins are of various nature and offer acomplex extracellular environment to the cells (similar to aphysiological tissue environment), 5) the tridimensional organization ofthe extracellular protein matrix is also similar to the physiologicaltissue environment. Physically: 1) tissue sheet are thick (˜50 to >200μm) compared to a monolayer (˜5 μm thick) and are easily visible to thenaked eye, 2) they can be peeled off a culture substrate with regulartweezers, 3) they are peeled off the culture substrate as one single,intact sheet covering the entire culture surface and containingpractically all the cells of the culture and the extracellular matrixproteins produced by the cells, 4) these sheets are robust enough thatthey can be easily manipulated with common surgical instruments, 5)these robust tissue sheets have shown a resistance to puncture exceedinga 800 gmf applied with a 8 mm spherical piston. To produce these tissuesheets, a cell population (homogenous or heterogeneous) is cultured inthe presence of ascorbic acid, or one of its derivatives, as well asother commonly available culture media components, to promoteextracellular matrix protein production. After an extended cultureperiod, enough extracellular matrix protein is produced to make acoherent tissue sheet. These tissue sheets are now an integral part ofthe new field of tissue engineering, term sheet-based tissueengineering, and have been used to create skin, blood vessels,myocardial patches, vascular patches, heart valves, and soon, morecomplex organs. This novel technology allows for the first time theproduction of mechanically sound living tissues and organs, that can bemade exclusively from these living tissue sheets without the need forany exogenous structural component such as synthetic scaffolds oranimal/cadaver derived products. Furthermore, sheet-based tissueengineering opens the door to the production or tissues and organs madefrom a patients own cells, avoiding all rejection complications.

The sheet-based tissue engineering methods and compositions describedherein utilize cells that are obtained and cultured in vitro into arobust sheet of cells. These sheets demonstrate puncture strength ofabout 50–100 grams force (gmf), typically about 100–300 gmf, more oftenabout 300–800 gmf, but most often >800 gmf, a mechanical feature notfound in any other tissue engineering or cell culture technology. Forexample, utilizing the techniques and methods described herein cells canbe extracted from a biopsy (typically from the same subject into whichthe cell sheet will ultimately be implanted) and expanded in vitro. Thecells can be genetically modified so that the genetically engineeredcells over-express, for example, an anti-restenotic or anti-thrombogenicagent or an agent that increases the physical compliance of the cells tophysiological stress. The unmodified or genetically modified cells arethen grown into a robust tissue sheet which can then be used to form atubular structure or which can be wrapped around a biocompatible stentto form a tissue-wrapped stent. The culturing of cells into a robusttissue sheet allows for a much higher density of cells to be deliveredand, when the cells are genetically modified, to deliver a higher dosingrange of, e.g., a therapeutic agent. The robust sheet of cells providesa fundamental difference/advantage over the current state of the art.For example, (1) the robust sheet allows for a much higher density ofcells to be delivered and therefore higher dosing of therapeutic agents;(2) the robust sheet acts as a membrane such that blood does not leakthrough and cells cannot migrate through; (3) the robust sheet can beused to envelope a stent, thus delaying foreign body responses; and (4)the robust sheet can be endothelialized to provide an anti-thrombogenicsurface on the lumen of a living stent or tissue-wrapped stent.

A tissue-wrapped stent (e.g., a robust sheet of cells wrapped about astent) can also expand with the stent so that a significant portion ofthe cells are still living after stent placement. In one aspect, thesheet provides a coating for the synthetic stent such that the syntheticmaterial of the biocompatible stent is “hidden” from the immune system.In addition, the coating of a biocompatible stent with the tissue sheetprovides a significant barrier against cell migration/proliferation intothe lumen of the stent thereby reducing the risk of intimal hyperplasia.

In another aspect, a tissue sheet of the invention is substantiallydecellularized to provide extracellular matrix materials provided by thepopulation of cells. In some cases, it may be advantageous todecellularize or denature all or part of the tissue engineeredconstruct. A decellularized sheet may have a reduced level ofimmunogenicity, and may provide a better matrix for endothelial seedingon the lumen, since the endothelial cells do not have to compete withthe more proliferative or more robust cell types. Decellularizing ordenaturing the sheet may also enhance the mechanical characteristics ofthe construct. The sheets, or the tissue within the construct, may bedecellularized, denatured, or chemically modified using a variety oftechniques. In the simplest embodiment, the sheet can be air-dried orlyopholized to kill the cells. Thermal shock, acoustic treatment,changes in pH, osmotic shock, mechanical disruption, or addition oftoxins can also induce cell death or apoptosis. Similarly, the sheet canbe crosslinked or fixed using agents such as paraformaldahyde. Othertreatments to decellularize or denature the tissue are possible usingradiation, detergents (SDS or triton ×100), enzymes (RNAase, DNAase), orsolvents (alcohol, acetone, or chloroform). These techniques are onlysome of the examples of techniques to decellularize, denature orchemically modify all or part of the tissue and are not meant to limitthe scope of the invention. For example, methods of decellularizing canutilize, for example, enzymes such as lipases combined with otherenzymes and, optionally, detergents. See, for example, WO 9603093A andWO 9632905A, incorporated herein by reference. Treatment with hypotonicand/or hypertonic solutions, which have nonphysiological ionicstrengths, can promote the decellularization process. These variousdecellularization solutions generally are suitable as treatmentsolutions. Proteases also can be used effectively to decellularizetissue. The decellularization can be performed in stages with some orall of the stages involving differential treatments. For example, apotent mixture of proteases, nucleases and phospholipases could be usedin high concentrations to decellularize the tissue. The decellularizedextracellular matrix may then have applied another tissue sheet oranother decellularized sheet. For example, one can roll a living layeron top of a decellularized layer.

Sheet-based tissue engineering, and in particular a tubular sheet ofcells or the tissue-wrapped stent described herein are different fromprior tissue engineering art. Prior methods of generatingtissue-engineered grafts had three fundamental aspects. First, priortissue engineering grafts require the use of a three dimensionalscaffold (such as a porous polymer or fabric) to provide mechanicalsupport for the cells. Second, prior tissue culture techniques would notresult in a robust tissue sheet that can be handled surgically. In fact,after a few weeks in culture, most cell culture techniques result inspontaneous detachment of the cells and loss of the culture. Third,prior tissue-engineering techniques relied heavily on allogeneic cellsources thereby inducing immune and/or inflammatory responses uponimplantation into a subject, which promotes infiltration, andproliferation into the lumen of a vessel or stent, the primary long-termfailure mode of other stenting technologies.

In one aspect, the invention provides a tissue-wrapped stent technologythat addresses both the short-term and long-term failures as describedabove. As one beneficial aspect there is provided techniques to obtaincells from a subject, genetically modify the cells to over-express ananti-restenotic or anti-thrombogenic agent and then grow these modifiedcells into a robust tissue sheet. Examples of anti-restenotic oranti-thrombogenic agents include nitric oxide synthase, PGI2(cyclooxygenase-1), tissue inhibitor of metalloproteinase-1 (or otherMMP inhibitors), tissue plasminogen activator (or other thrombolyticagents), heparin and derivatives thereof, tissue factor pathwayinhibitor (or other anti-inflammatory agents), and statins. The cellsmay also be modified to express anti-proliferation/activation productsthat can be triggered or suppressed (to control potential intimalhyperplasia) including, for example, retinoblastoma family of genes, E2Fdecoy, AP-1 decoy, cyclin-dependent kinase inhibitors, I kappa B alpha,and the like. During growth in culture the cells may be cultured withagents that promote cellular proliferation and growth. Such agentsinclude a number of growth factors that can be selected based upon thetissue to be grown and the cell types present (e.g., keratinocyte growthfactor (KGF); vascular endothelial cell growth factor (VEGF); plateletderived growth factor (PDGF); fibroblast growth factor (FGF); atransforming growth factor (TGF) alpha, beta, and the like; insulin;growth hormone; somatomedins; colony stimulating factors;erythropoietin; epidermal growth factor; hepatic erythropoietic factor(hepatopoietin); and liver-cell growth factor to name a few, others areknown in the art). Serum, such as fetal bovine serum (FBS) or the like,can also provide some of these growth factors. In addition, agents suchas ascorbic acid can be used to increase extracellular matrixproduction.

A tissue manipulation device (e.g., one or more such devices such ascontrol rods) is used to secure a growing tissue sheet to a flask orother culture container and prevent spontaneous detachment. Moreover,the tissue manipulation device (e.g., control rods) allow for easiersheet handling while a tissue sheet is being wrapped around a stent orin the formation of a tubular structure. A tissue manipulation devicemay be added to a culture container before seeding with the cells. Insome aspects of the invention a tissue manipulation device may be addedafter seeding a culture container with a population of cells (e.g.,immediately after seeding the culture container or may passages or weeksafter seeding the culture container). For example, tissue sheetstypically begin to spontaneously detached after being in culture for aprolonged period of time, in one aspect of the invention, a tissuemanipulation device is added to culture just prior to or during thatstage of spontaneous detachment in order to further facilitate prolongedculture and development of a robust tissue sheet.

A tissue manipulation device (e.g., control rods) are made fromrelatively inert biocompatible materials (including, but not limited to,Teflon®, magnetic material, a magnetizable material, a polypropylene, asteel (stainless steel) or a steel alloy, a titanium or a titaniumalloy, a polystyrene, a glass, and any combination thereof). In anotheraspect, one or more tissue manipulation device(s)s are temporarilyintegrated into a robust tissue sheet during the growth andproliferation phase of cells in culture. During this phase, cells areseeded onto a cell growth substrate in a culture container and the cellsgrow and envelop a tissue manipulation device (e.g., one or more controlrods). As the cells and their corresponding matrix proteins grow and aredeposited, respectively, during maturation into a tissue sheet, thecells grow onto or adhere to and envelop a tissue manipulation device(e.g., one or more control rods). In order to assemble these sheets intoa more complex three-dimensional tissue, the tissue manipulationdevice(s) can be used to detach the tissue sheet and support it duringassembly and manipulation (FIG. 1). The control rods can also be used tomagnetically secure or manipulate the sheets using external magnets andferrous containing rods. For example, a ferrous tissue manipulationdevice coated with Teflon can be used to hold a tissue to a plate bymagnets placed on the external surface of a culture container.

By “cell growth substrate” means any number of materials andcompositions, many of which are currently commercially available,including for example, a tissue culture plate of a rectangular orcircular shape having a growth area of about 1 cm² to greater than 500cm², multiwell tissue culture plates having 2 wells to more than 90wells per tissue culture plate, and tissue specimen slides as used inmicroscopic analysis. Other cell growth surfaces and devices are readilyapparent to those of skill in the art. The methods and apparatus of theinvention can be used with any of the foregoing cell or tissue cultureplates, slides and devices.

A tissue sheet can be removed from culture utilizing a tissuemanipulation device and can be further manipulated using the tissuemanipulation device as needed to give rise to a desired organ orstructure. Once the tissue sheet has been shaped into the appropriateorgan or structure, the tissue manipulation device can be removed fromthe sheet. By making the tissue manipulation device from a non-adherentmaterial or a material that cells bind to only weakly, such as stainlesssteel or ePTFE, the tissue manipulation device can be removed withoutdamaging the tissue sheet. In other applications, it is conceivable thatstronger adhesion would be desirable in which case different materialssuch as treated polystyrene is used. In yet other applications of thetechnology biodegradable materials may be desirable including, forexample, polylactic acid, polyglycolic acid, collagen based material,cat gut sutures, and the like. Typically the tissue manipulation devicesare substantially non-porous or non-porous thereby making it easier toremove the devices when needed.

Additionally, as discussed elsewhere herein, the integrated tissuemanipulation device can also be used to increase/promote cell adhesionand/or the formation of a tissue sheet in a culture container.Accordingly, the invention provides methods to simplify the manipulationof films or membranes of living tissues as required in manytissue-engineering techniques and in the formation of tissue sheet-basedvascular grafts and tissue sheet-wrapped stents. In general, the tissuemanipulation devices (e.g., rods) are introduced into a cell culturesuch that they become integrated into a tissue or are in contact (e.g.,not intergrated into the tissue) with the tissue comprising a populationof cells as the cells proliferate. The tissue manipulation devicesshould be mechanically robust enough to support a tissue during normalhandling, tissue manipulation and/or tissue engineering techniques, ortest procedures.

Typically a tissue manipulation device will comprise a material that canbe sterilized by conventional techniques such as heat, ethylene oxide,or gamma sterilization. The tissue manipulation device should be of abiocompatible material in order to prevent cytotoxic effects upon cells.Depending upon the desired culture conditions, tissue construct to bemade, and cell types present, the tissue manipulation device may be madeof either a biodegradable or a non-biodegradable material. For example,where the tissue construct comprises a three-dimensional structure thatprevents easy removal of the tissue manipulation devices or removal ofthe tissue manipulation device would result in undesirable damage to atissue sheet, then the tissue manipulation devices should be made of abiodegradable material.

In one particularly embodiment, at least two tissue manipulation devices(e.g., 2, 3, 4, . . . , or more than 10) comprising rods are made from316 stainless steel wires approximately 0.050 inches in diameter. Therods are bent to the shape of the cell culture container, then steamsterilized. Cells are seeded in a culture container comprising thetissue manipulation devices and allowed to grow to a confluent monolayeror tissue sheet. The tissue manipulation rods are position and securedusing magnets placed on the outside of a culture container (e.g., aflask) to act as clamps. The tissue manipulation rods are then left inplace until such time as the cells of a tissue sheet substantiallyenvelop the tissue manipulation devices. Cells of the tissue grow ontoand envelop the stainless steel and only weakly adhere therebyintegrating the tissue manipulation rods into the tissue sheet. When thetissue sheet reaches a desired mechanical characteristic, the tissuesheet and tissue manipulation device combination can be peeled away fromthe original cell culture substrate once the magnetic clamps areremoved. The tissue manipulation devices can now be used to manipulatethe tissue. For example, where a desired tissue construct comprises avascular graft such as a blood vessel or tissue-wrapped stent, thetissue manipulation devices can be loaded onto a rotating mandrel suchthat the sheet can be fed into an automatic rolling device. Likewise,the sheets can be more easily and reproducible handled for the assemblyof other tissue engineered organs like skin, livers, ligaments or heartvalves where it is necessary to arrange sheets or segments of sheetsinto stacks.

The tissue manipulation device can be withdrawn from a cell sheet ortissue quite easily when the tissue manipulation device comprises arelatively inert material such as stainless steel, untreated Delrin™, orePTFE. In some applications, it may be desirable to adhere the tissuemanipulation devices to a cell sheet or tissue. For example, where atissue will be used for mechanical testing it is not necessary to removethe tissue manipulation devices, so a wider range materials, includingadherent materials can be used. In other aspects of the invention, whereheart valves or temporary support livers (e.g., extracorporeal liverdevices), the tissue manipulation devices may be a permanent fixturethat also allows a wider range of materials to be used. Wherebiodegradable-biocompatible materials are used the materials should berobust enough in culture to help with the manipulation of the tissuesheet, but once manipulated and/or implanted would be resorbed by thecells or body. In another aspect, the tissue manipulation device maysimply be cut away from the tissue sheet once it is no longer needed.

A number of cell types may be cultured on a cell growth substrate usingstandard cell culturing techniques. Typically the cells used in themethods and systems of the invention comprise adherent cell types. Suchcells when cultured under appropriate conditions divide and expand tocover and adhere to a cell growth substrate. The cell types may beallogeneic or autologous with respect to a subject to which that atissue construct derived from the cells is ultimately implanted or used.

Ideally, cells are harvested from an autologous source, expanded andmanipulated in culture, then re-implanted into the donor (see, e.g.,U.S. Pat. Nos. 5,512,475; 5,780,299; and 5,866,420, which areincorporated herein by reference in their entirety). However, currenttissue engineering strategies use a common technique, which is toculture the cells within a synthetic scaffold that provide requisitemechanical strength. These scaffolds are typically based on eitherresorbable polymers or porous meshes of biological materials such ascollagen foams.

The methods and systems provided herein do not require synthetic cellscaffolds and are based in part upon the pioneering work of Auger andL'Heureux U.S. Pat. No. 5,618,718 and L'Heureux and McAllister U.S.patent application Ser. No. 09/444,520, the disclosures of which areincorporated herein by reference.

As one beneficial aspect of the invention there is provided techniquesto culture a subject's own cells into a robust sheet. The inventionprovides compositions and methods to generate sheets of autologousand/or allogenic cells. These cell sheets are robust and can be shapedor molded into a variety of organs, including blood vessels, heartvalves, and the like. The cells in the sheet can be manipulated into adesired shape or structure prior to or during implantation to assist inthe replacement of organs or to assist in the reconstruction and/orhealing of an existing organ.

Additionally, the tissue manipulation device can act as a supportstructure for more complex three dimensional structures such as heartvalves. Prior art describes various techniques to suture or gluexenogenic tissue such as bovine pericardium to a heart valve frame,however, these technologies do not describe a technique to produce anautologous heart valve formed by mounting a robust sheet of human cellsas described herein onto a heart valve frame work. There are a varietyof possibilities envisioned. In one embodiment, the autologous humansheet is grown directly onto the heart valve frame work such that thetissue adheres to the framework during the culture process. This wouldlikely require a non-planar culture dish that would allow the formationof an annular ring with tissue that forms the valve leaflets. In asimilar configuration, the sheet can be grown onto the tissuemanipulation framework in a planar configuration and then folded into athree dimensional configuration suitable for use as a heart valve. Inboth cases, the tissue sheet can be grown as described herein, detachedfrom the cell culture substrate, and then attached to the valve framework by chemical or mechanical means as is known in the art. Thisincludes, but is not limited to, sewing, suturing, gluing, hooking,clamping, riveting etc. In all configurations, this invention describesa technique to produce a novel heart valve where the leaflets of thevalve are formed by an autologous tissue engineered sheet.

As discussed above a tissue sheet may comprise any number of variouscell types. Stromal cells including, for example, fibroblasts may bederived from a number of organs including, for example, the skin,pancreas, liver, and the like. Samples from which the fibroblasts arederived include, for example, from a biopsy (where appropriate), from acadaver, or from disposed organ tissue. The fibroblast may be derivedfrom the same organ for which they will be ultimately used to develop atissue-engineered organ in vitro (e.g., derived from cardiovasculartissue for development of a vascular graft). Typically the fibroblastwill be obtained from the same individual and later reimplanted (i.e.,autologously). Precursor cells (e.g., stem cells of a particular tissue)that are not fully differentiated or that lack a particular MHC may beused. Such cells have the advantage of not inducing an immune responsedue to recognition of a fully differentiated cell as being “non-self”.There are a number of stem cells and precursor cells known in the art(e.g., mesenchymal stem cells, hematopoietic stem cells, circulatingstem cells, endothelial stem cells, embryonic germ cells, and the like),which are applicable to the culturing techniques of the invention solong as the cells are adherent or differentiated to adherent cellsduring culturing.

Stromal cells (e.g., fibroblasts) may be obtained by treatment of anappropriate organ or tissue that is to serve as the source of thestromal cells. Techniques for treatment of an organ or tissue to obtainstromal cells are known to those skilled in the art (see, e.g.,Freshney, Culture of Animal Cells. A Manual of Basic Technique, 2d Ed.,A. R. Liss, Inc., New York, 1987, Ch. 9, pp. 107–126). For example, thetissue or organ can be mechanically disrupted and/or treated withdigestive enzymes or chelating agents to weaken the interactions betweencells making it possible to obtain a suspension of individual cells.Typically the method will include a combination of mechanicaldisruption, enzyme treatment and chelating agents. In one technique thetissue or organ is minced and treated simultaneously or subsequentlywith any of a number of digestive enzymes either alone or incombination. Examples of enzymes useful in dissociating cells include,but are not limited to, trypsin, chymotrypsin, collagenase, elastase,hyaluronidase, DNase, pronase, dispase, and the like. Mechanicaldisruption can also be accomplished by, for example, the use ofblenders, sieves, homogenizers, pressure cells, and the like.

The resulting suspension of cells and cell clusters can be furtherdivided into populations of substantially homogenous cell types. Thiscan be accomplished using standard techniques for cell separationincluding, for example, positive selection methods (e.g., clonalexpansion and selection of specific cell types), negative selection(e.g., lysis of unwanted cells), separation based upon specific gravityin a density solution, differential adherence properties of the cells inthe mixed population, fluorescent activated cell sorting (FACS), and thelike. Other methods of selection and separation are known in the art(see, e.g., Freshney, Culture of Animal Cells. A Manual of BasicTechniques, 2d Ed., A. R. Liss, Inc., New York, 1987, Ch. 11 and 12, pp.137–168).

One specific method of isolating stromal cells (e.g., fibroblasts)includes the mincing of a tissue in Hank's Balanced Salt Solution (HBSS)or other similar solution. The tissue is then incubated in a solution oftrypsin under conditions and for a time sufficient to separate the cells(e.g., at about 4° C. for 1 to 12 hours). The separated cells aretypically suspended in a high protein medium (e.g., media with fetalbovine serum or human serum (including autologous serum)), pelleted bycentrifugation and plated onto tissue culture plates. Fibroblasts, forexample, typically attach to the tissue culture plastic before othercells, thereby giving rise to a population of fibroblast cells. Theresulting population of fibroblasts cells are typically substantiallyhomogenous, but may contain additional cell types including macrophages,endothelial cells, epithelial cells, and the like, present in the tissuefrom which the fibroblasts are isolated. The fibroblasts are then grownto confluence in the presence of a tissue manipulation device (e.g., arod of biocompatible material), which facilitates growth of a robustsheet and/or removal of the confluent fibroblast culture (e.g., a tissuesheet of fibroblasts) and manipulation of the confluent tissue sheet ofcells to develop a tissue construct.

A sheet grown from fibroblast or other cell types are grown for periodsof days to weeks in a culture container. Typically the cell sheets aregrown for about 1–24 weeks, depending on the application, donor age,viability of the particular cell population, and the like. A culturecontainer includes any number of materials and compositions, many ofwhich are currently commercially available, including for example, atissue culture plate of a rectangular or circular shape having a growtharea of about 1 cm² to greater than 500 cm², multiwell tissue cultureplates having 2 wells to more than 90 wells per tissue culture plate,and tissue specimen slides as used in microscopic analysis. Other cellgrowth surfaces and devices are readily apparent to those of skill inthe art. The methods and apparatus of the invention can be used with anyof the foregoing cell or tissue culture plates, slides and devices.

In most cell culture applications, adherent cell cultures can only bemaintained for a few days to a few weeks before the individual cellsrelease from a substrate. The addition of agents that promote cellgrowth, viability and/or adhesion can be used during the cultureprocess. For example, addition of agents such as ascorbic acid, retanoicacid, and copper can be used to increase the production of extracellularmatrix proteins thereby generating a more robust tissue sheet of cells.Moreover, by treating the cell culture surface/substrate withextracellular matrix proteins or other factors (e.g., a protein such asgelatin or fibrin), adhesion can be prolonged. The introduction of oneor more tissue manipulation device(s)s (e.g., a plurality of controlrods), such as those described herein, can be used to hold down theedges of the sheet on a culture container or substrate thus preventingspontaneous sheet detachment. The rods or tissue manipulation device canbe designed such that they generate significant clamping forces (e.g.,via gravity, magnetic forces and the like) to effectively secure thesheet. Moreover, the rods can be made of a biocompatible material suchas ePTFE (e.g., a ePTFE outer surface and a metallic core) or stainlesssteel that will be slightly adherent to the cells present in a tissuesheet. In this manner, once a tissue sheet is removed from the culturesurface it can be handled easily. This is an important development forboth design and manufacturing that has not been described previously.

In another aspect, a stent-less tubular construct can be made comprisingcells grown to a robust tissue sheet. In this aspect of the invention,the tissue sheet is removed from culture and manipulated using one ormore tissue manipulation device(s)s that have been integrated throughcellular growth into the tissue sheet. The stent-less tubular constructis mechanically formed by creating a cylindrical structure comprisingthe tissue sheet. The cylindrical structure will comprise one or morelayers or cells and/or one or more layers of the tissue sheetperpendicular to the radius of the cylindrical tubular structure.

In one aspect of forming the tubular structure the tissue sheet ismanipulated around a tubular mold or mandrel and then the tubular moldor mandrel is removed. For example, once a tissue sheet of cells isobtained, it can be rolled on the outer surface of a tubular support(e.g., a mold) of varying diameter to give it a tubular form. If thesheet is held in place while rolled up for a few hours in suitable cellculture conditions, it will adhere to itself relatively firmly and willstay in its tubular form, around its tubular mold. Alternatively, theends may be sewed or glued to one another to assist in the formation ofa tubular structure. The tubular tissue hence created may be kept inculture conditions for extended periods of time and submitted to variousculture conditions to influence its ongoing development (ascorbic acid,serum, mitogens, and the like). The tubular mold is typically made of a“non-sticky” material thereby not allowing adhesion between the cellsheet and the material in order to facilitate removal of the mold fromthe lumen of the tissue sheet. Examples of such “non-sticky” materialinclude ePTFE, polystyrene, stainless steel, and the like. At the end ofa maturation period, the tubular tissue can be slid off its tubular moldto provide the basic scaffold for the construction of a more complextissue culture system. For example, endothelial cells can be seeded onthe inner surface of a tubular tissue sheet and/or smooth muscle cellson the exterior surface, hence producing a basic blood vessel constructor prosthesis. Such a tubular tissue prosthesis can be useful in tissuetransplantation as it can be made from the cells of the graft recipient,thus circumventing immunological rejection of the prosthesis.Furthermore, the tubular tissue can be rolled up in a similarly producedsheet to obtain a multi-layer tubular tissue sheet. Cells which formadditional layers can be of identical nature to the cells of a firstlayer to obtain a thicker tissue, or of different origin (such asendothelial, smooth muscle cells, and the like).

In addition to fibroblasts, other cells may be added to tissue sheeteither before or after removal from culture. Such additional cellscomprise parenchymal cells such as smooth muscle cells and/orendothelial cells. Sprinkling the sheet with endothelial cells, forexample, helps to grow vascular networks (vasovasorum) within the wallof the rolled vessel. In normal vascular tissue cell layers formcreating distinct areas of a blood vessel. Endothelial cells are foundin the highest number in the lumen of the blood vessel and smooth musclecells present on the external surface of the blood vessel. Fibroblastsand other stromal cells are present between the endothelial and smoothmuscle cells. Accordingly, the seeding of a tissue sheet can performedsuch that the endothelial, fibroblast and smooth muscle cell layers arepresent in the vascular graft. For example, fibroblasts may be culturedto form a robust tissue sheet of cells, the tissue sheet is then seededwith endothelial cells whereupon manipulation of the tissue sheetcomprising the endothelial cells into a tubular structure results in theendothelial cells being present on the lumen surface of the tubularstructure. Smooth muscle cells can then be seeded on the externalsurface of the tubular structure. The tissue sheet-based construct canthen be implanted or further cultured for a time sufficient to allow thecells to interact and grow. Such culture conditions may includeculturing the tubular construct in a bioreactor that generates flow andshear stresses typically present in a vascular tissue. A bioreactorsimilar to that described in U.S. Pat. No. 6,121,042 (the disclosure ofwhich is incorporated herein by reference) may be used.

In another aspect of the invention, a tissue-wrapped stent can begenerated. In this aspect, a tissue sheet is manipulated around (e.g.,to surround one or more times) a biocompatible and/or biodegradablematerial (e.g., a stent) having a lumen, and the lumen is then seededwith endothelial cells and/or smooth muscle cells. Alternatively,endothelial cells and/or smooth muscle cells may be added to thebiocompatible and/or biodegradable material prior to adding thefibroblast sheet. In one aspect of the invention a biocompatible stentis used as a support for forming the tubular structure. Thebiocompatible stent is mounted on a temporary support mandrel and thetissue sheet is then layered one or more times around the biocompatiblestent. Cells including, for example, endothelial cells, pericytes,macrophages, monocytes, plasma cells, mast cells, adipocytes, and thelike, may be added independently or together. The biocompatible and/orbiodegradable material typically will be substantially non-porous (e.g.,lacking interstitial spaces) and where such pores are present they areof a size that does not allow for diffusion of the cell typesinto/through the material. Examples of pores that are sufficiently smallrange in size from 10 μm to 100 μm. In one aspect, the biocompatiblematerial is a “non-sticky” material thereby not allowing adhesionbetween the cell sheet and the material. Examples of such “non-sticky”material include ePTFE, polystyrene, stainless steel, and the like.

The sheet-wrapped stent has many advantages that distinguish it fromother covered stents. These include: (a) the stent can be coated with asubject's own cells (e.g., fibroblasts, endothelial cells, and/or smoothmuscle cells) which will slow the foreign body response and subsequentfailures; (b) where the subject's cells are genetically modified, suchcells and their progeny will continue to produce a therapeutic and/ordiagnostic agent (e.g., a polypeptide or polynucleotide); (c) a tissueengineered vascular graft comprising a sheet of cells, unlike simplecell seeded stents, provides a barrier to migrating cells and subsequentintimal hyperplasia; (d) the sheet can be seeded with functionalendothelial cells, thus providing a uniform lumen with ananti-thrombogenic surface; and (e) a sheet of cells can be assembledinto a tubular construct that can act as a stand alone stent or a standalone bypass conduit.

In one aspect, the invention provides a process by which a stent iswrapped in a sheet of autologous or allogenic cells to provide atissue-wrapped stent. A stent refers to any device capable of beingplaced into contact with a portion of a wall of a lumen of an artery,vessel, or other tubular organ structure. Stents useful in the methodsand compositions of the invention include classical stents used inintravascular applications, as well as any prosthesis that may beinserted and held where desired in a lumen.

Typically, a stent has a lumen wall-contacting surface and alumen-exposed surface. A stent is shaped generally as a tubularstructure but includes discontinuous tube structures. A lumen-wallcontacting surface is the outside surface of the stent and thelumen-exposed surface is the inner surface of the stent. The stent caninclude polymeric elements, metallic elements, filamentary elements, orcombinations thereof. In one aspect the surfaces (e.g., the lumen-wallcontacting surface and/or the lumen exposed surface is coated withextracellular matrix material (e.g., fibronectin, fibrin, collagen, orother extracellular matrix material) prior to contact with a tissuesheet in order to promote adherence of the tissue sheet to the stent.

In one aspect a deformable metal wire stent is used in conjunction withthe tissue sheet. An example of a metal wire stent is described in U.S.Pat. No. 4,886,062. Other metallic stents useful in this inventioninclude those described in U.S. Pat. No. 4,733,655 and U.S. Pat. No.4,800,882. Polymeric stents can also be used in this invention andinclude stents comprising biodegradable and non-biodegradable materials.

Stents useful in the invention generally comprise a biocompatiblematerial. In some aspect the stent is coated with a drug that assists inreducing restenosis or for the delivery of a therapeutic agent. Suchdrug-eluting stents are also useful in the methods and compositions ofthe invention in combination with both genetically engineered tissuesheets as well as tissue sheets comprising cells that have not beengenetically engineered. Generally stent designs include those describedin U.S. Pat. Nos. 4,733,655, 4,800,882, and 4,886,062. Such designsinclude both metal and polymeric stents, as well as self-expanding andballoon-expandable stents. Examples of drug-eluting stents include thosedescribed in U.S. Pat. No. 5,102,417 and in International PatentApplication Nos. WO 91/12779 and WO 90/13332, (the disclosures of whichare incorporated herein).

In one aspect, the invention provides a cell-based sheet wrapped stenttechnology. As one beneficial aspect there is provided techniques toculture a subject's own cells into a robust sheet. These robust sheetsof cells can then be applied to stents. Once a sheet of cells isobtained, it can be rolled on the outer surface of a tubular support ofvarying diameter to give it a tubular form. If the sheet is held inplace while rolled up for a few hours in suitable cell cultureconditions, it will adhere to itself relatively firmly and will stay inits tubular form, around its tubular support. The tubular tissue hencecreated may be kept in culture conditions for extended periods of timeand submitted to various culture conditions to influence its ongoingdevelopment (ascorbic acid, serum, mitogens, and the like).

In one aspect the tubular support may comprise an anti-thrombotic and/oranti-restenosis agents including proteins, polypeptide, peptides,peptidomimetics, and small molecules. One particular advantage is thatthe robust sheet of cells slowly replaces that stent material as theanti-thrombotic agents degrade or are used up. By this technique thesubject's own cells are allowed to replace and/or reconstruct thedamaged vasculature. Anticoagulant substances such as heparin andthrombolytic agents have also been incorporated into a stent, asdisclosed, for example, in U.S. Pat. Nos. 5,419,760 and 5,429,634.

A particular advantage of the tissue wrapped-stents of the inventioncompared to a seeded cell stent is that the robust tissue sheet of cellsprovides an immediate barrier that prevents infiltration of extraluminalcells. In contrast, cell seeded stents do not provide the same barrierfunction and thus extraluminal cells are capable of infiltrating thestent and the lumen of the sent thereby leading to intimal hyperplasia.In order to promote the barrier function of the sheet-wrapped stents,the tissue sheet may be cultured for a time sufficient and undersufficient conditions to allow fusion between the cells of the tissuesheet and/or the stent. In some aspects, the sheet would have a shortermaturation time after wrapping onto the stent so that the stent can beexpanded within the sheet while the sheet simply unwinds slightly. Inanother aspect, the stent material is biodegradable or may be entirelyabsent. During the culture time, the tissue construct can be seeded withadditional cell types (e.g., endothelial cells and/or smooth musclecells) either autologous or allogeneic. The addition of such additionalcell types can assist to reduce the likelihood of thrombosis.

In one aspect, the invention provides a cell-based sheet wrapped stenttechnology. As one beneficial aspect there is provided techniques toculture a subject's own cells into a robust sheet. These robust sheetsof cells can then be applied to stents. Once a sheet of cells isobtained, it can be rolled on the outer surface of a tubular support ofvarying diameter to give it a tubular form. If the sheet is held inplace while rolled up for a few hours in suitable cell cultureconditions, it will adhere to itself relatively firmly and will stay inits tubular form, around its tubular support. The tubular tissue hencecreated may be kept in culture conditions for extended periods of timeand submitted to various culture conditions to influence its ongoingdevelopment (ascorbic acid, serum, mitogens, etc.).

In one aspect the tubular support may comprise an anti-thrombotic and/oranti-restenosis agents including proteins, polypeptide, peptides,peptidomimetics, and small molecules. One particular advantage is thatthe robust sheet of cells slowly replaces that stent material as theanti-thrombotic agents degrade or are used up. By this technique thesubject's own cells are allowed to replace and/or reconstruct thedamaged vasculature.

In yet another aspect of the invention there is provided an expandablestent that is covered by a tissue sheet of autologous and/or allogenicfibroblasts to form a tissue construct. In one aspect, the fibroblastsare derived from fibroblast precursor cells (e.g., mesenchymal stemcells, hematopoietic stem cells, circulating stem cells, and the like)and thus typically lack the antigenic determinants that would beindicative of a cell derived from a particular individual. Thefibroblasts or precursor cells may be genetically modified to express orover express polypeptides or polynucleotides that prevent or retardrestenosis, thrombosis, and/or reduce inflammation. Such a tissueconstruct may include the seeding of autologous endothelial cells withinthe lumen of the stent to reduce thrombosis.

In another aspect of the invention, the cells of a tissue sheet can begenetically modified to express diagnostic and/or therapeutic product(e.g., a polypeptide or polynucleotide) to assist in tissue healing,replacement, maintenance, diagnosis, and the like of an implanted tissuesheet. Unlike the drug eluting coatings, however, these cells, and theirprogeny, can be engineered to continually produce a desired geneproduct. In another aspect the cells may only transiently produce aproduct. Methods of permanent and transient transfections are known andwill be described further herein. One particular advantage of utilizinga tissue sheet comprising genetically engineered cells is that a largeamount of cells may be utilized compared to simply seeding a relativelyfew number of genetically engineered cells.

The cells present in a tissue sheet may be genetically modified toproduce a diagnostic or therapeutic product. By diagnostic product ismeant a product that is expressed due to a genetic transformation of thegenotype of a cell wherein the product assists in the diagnosis of, forexample, proper stent or tubular tissue placement or the failure of astent or tubular tissue. A diagnostic product can be a polypeptide,peptide, polynucleotide, or a by-product of a reaction resulting fromexpression of a product comprising an enzyme. For example, as shearstress increases in an implanted stent due to intimal hyperplasia,specific promoters can be activated to express a marker indicative ofstent failure. By a therapeutic product is meant a product that isexpressed due to a genetic transformation of the genotype of a cellwherein the product assists in the treatment and/or patentcy of a stentor tubular tissue. The therapeutic product may be a polypeptide,peptide, peptidomimetic, soluble ligands (e.g., homooligomers,heterooligomers), and/or polynucleotides (e.g., antisense or ribozymemolecules). Examples of therapeutic products include anti-inflammatoryfactors such as anti-GM-CSF, anti-TNF, anti-IL-1, anti-IL-2, and thelike. Alternatively, the cells in the tissue sheet may be geneticallymodified to inhibit expression of polypeptide or polynucleotide thatpromotes inflammation, e.g., GM-CSF, TNF, IL-1, IL-2, or inhibitexpression of MHC in order to lower the risk of tissue rejection. As yetanother aspect, the cells of the tissue sheet can be geneticallymodified to block expression of a polypeptide or polynucleotide (e.g., aribozyme or antisense molecule) that causes smooth muscle cells toproliferate and migrate into the lumen of a stent of a vascular graft toprevent neointimal hyperplasia.

A polynucleotide or nucleic acid molecule refers to a polymeric form ofnucleotides at least 9 bases in length. An isolated polynucleotide is apolynucleotide that is not immediately contiguous with either of thecoding sequences with which it is immediately contiguous (one on the 5′end and one on the 3′ end) in the naturally occurring genome of theorganism from which it is derived. The term therefore includes, forexample, a recombinant DNA or RNA that is incorporated into a vector, aviral vector, or into the genome of a host cell such that the isolatedpolynucleotide is heterologous to the genome or location in the genomeof a host cell. The nucleotides present in a polynucleotide used fortransformation and/or transfection of a host cell can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single- and double-stranded forms ofnucleic acid molecules.

A polynucleotide may be any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions.

In addition, polynucleotide as used herein can also refer totriple-stranded regions comprising RNA or DNA or both RNA and DNA. Thestrands in such regions may be from the same molecule or from differentmolecules. The regions may include all of one or more of the molecules,but more typically involve only a region of some of the molecules. Oneof the molecules of a triple-helical region often is an oligonucleotide.

As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons are“polynucleotides” as that term is intended herein. Moreover, DNAs orRNAs comprising unusual bases, such as inosine, or modified bases, suchas tritylated bases, to name just two examples, are polynucleotides asthe term is used herein. As discussed more fully below such modifiedpolynucleotides may be coated on biocompatible stents where thepolynucleotides are taken up by cells that come into contact with thestent (see, e.g., U.S. Pat. No. 5,962,427, which is incorporated hereinin its entirety).

Cells present in a tissue sheet may be genetically engineered usingrecombinant DNA techniques to generate a recombinant host celltransformed or transfected with a polynucleotide that encodes a productof interest (e.g., a polypeptide or polynucleotide that provides adiagnostic and/or therapeutic benefit). Methods of transforming ortransfecting cells with exogenous polynucleotide such as a DNA moleculeare well known in the art and include techniques such ascalcium-phosphate- or DEAE-dextran-mediated transfection, protoplastfusion, electroporation, liposome mediated transfection, directmicroinjection and adenovirus infection (Sambrook, Fritsch and Maniatis,1989).

The most widely used method is transfection mediated by either calciumphosphate or DEAE-dextran. Depending on the cell type, up to 90% of apopulation of cultured cells can be transfected at any one time. Becauseof its high efficiency, transfection mediated by calcium phosphate orDEAE-dextran is the method of choice for experiments that requiretransient expression of the foreign DNA in large numbers of cells.Calcium phosphate-mediated transfection is also used to establish celllines that integrate copies of the foreign DNA, which are usuallyarranged in head-to-tail tandem arrays into the host cell genome.

Yet another method of transfection or transformation that can be used togenerate genetically engineered/genetically-modified cells includes thetechnique of electroporation. The application of brief, high-voltageelectric pulses to a variety of mammalian cells leads to the formationof nanometer-sized pores in the plasma membrane of the cell(s). DNA istaken directly into the cell cytoplasm either through these pores or asa consequence of the redistribution of membrane components thataccompanies closure of the pores. Electroporation can be extremelyefficient and can be used both for transient expression of clonedheterologous polynucleotides and for establishment of cell lines thatcarry integrated copies of a polynucleotide. Electroporation, incontrast to calcium phosphate-mediated transfection and protoplastfusion, frequently gives rise to cell lines that carry one, or at most afew, integrated copies of a heterologous polynucleotide.

Other methods that can be used include liposome transfection, whichinvolves encapsulation of DNA and RNA within liposomes, followed byfusion of the liposomes with the cell membrane. Direct microinjection ofa polynucleotide into nuclei has the advantage of not exposing thepolynucleotide to cellular compartments such as low-pH endosomes.Microinjection is also used as a method to establish lines of cells thatcarry integrated copies of a polynucleotide of interest.

The use of retroviral vectors and adenoviral vectors for celltransfection is known in the art. Adenovirus vector-mediated celltransfection has been reported for various cells (Stratford-Perricaudet,et al. 1992). A host cell of the invention is typically a eukaryoticstromal or parenchymal cell that can be grown in culture to form arobust tissue sheet. The host cell(s) may be autologous to the subjectultimately intended to receive the host cell(s) or may be allogeneic.

It should be noted that a polynucleotide used to transform or transfecta host cell need not encode a product. A polynucleotide useful in theinvention can be a promoter or enhancer element that is flanked byoligonucleotide sequences that are identical to regions in the host cellgenome. Upon introduction of the promoter or enhancer construct theoligonucleotides align with the host cell genome and the promoter orenhance sequence is thereby integrated into the host cell genome duringnormal cellular replication. Typically the promoter or enhancer elementwill be heterologous to the location of integration and results in themodified expression (e.g., the up regulation or down regulation) of agene sequence in the host cell genome. Where down regulation occurs theheterologous sequence disrupts normal expression of a gene. Examples ofpromoter/enhancer elements useful in the genetically engineeredtissue-wrapped stents and tissue sheet-based vascular grafts include anelastin or elastase I regulatory element. The deposition of elastin iscorrelated with specific physiological and developmental events indifferent tissues, including the vascular grafts. For example, indeveloping arteries, elastin deposition appears to be coordinated withchanges in arterial pressure and mechanical activity. The transductionmechanisms that link mechanical activity to elastin expression involvecell-surface receptors. Once elastin-synthesizing cells are attached toelastin through cell-surface receptors, the synthesis of additionalelastin and other matrix proteins may be influenced by exposure tostress or mechanical forces in the tissue.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used togenetically modify a host cell. The vector ordinarily carries areplication site, as well as marking sequences which are capable ofproviding phenotypic selection in transformed cells. In constructingsuitable expression plasmids, the termination sequences associated withheterologous polynucleotides are also introduced into the expressionvector downstream from the sequences to be expressed to providepolyadenylation of the mRNA and termination.

For example, expression vectors for use in transforming or transfectingcells ordinarily include an origin of replication, a promoter locatedupstream of the polynucleotide encoding a particular product to beexpressed, along with any necessary ribosome binding sites, RNA splicesites, polyadenylation site, and transcriptional terminator sequences.

For use in mammalian cells, the control functions of the expressionvectors are often derived from viral material. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, Cytomegalovirusand most frequently Simian Virus 40 (SV40). The early and late promotersof SV40 virus are particularly useful because both are obtained easilyfrom the virus as a fragment which also contains the SV40 viral originof replication. Smaller or larger SV40 fragments can also be used,provided there is included the approximately 250 bp sequence extendingfrom the HindIII site toward the BglI site located in the viral originof replication. Further, it is also possible, and often desirable, toutilize promoter or control elements normally associated with thedesired polynucleotide encoding the product to be expressed, providedsuch control sequences are compatible with the host cell systems.

An origin of replication can be provided by construction of the vectorto include an exogenous origin, such as can be derived from SV40 orother viral (e.g., Polyoma, Adeno, VSV, BPV, CMV, rapalogue) source, orcan be provided by the host cell chromosomal replication mechanism. Ifthe vector is integrated into the host cell chromosome, the latter isoften sufficient.

The genetically engineered tissue-wrapped stents and tissue sheet-basedvascular grafts of the invention provide a method for introducingtherapeutic products not only locally (e.g., to prevent intimalhyperplasia and restenosis) but also systemically to treat disease anddisorders in a subject. In one aspect, the invention provides a suitablemeans for providing delivery of a therapeutic or diagnostic productand/or gene therapy. The usefulness of the genetically engineeredtissue-wrapped stents and tissue sheet-based vascular grafts arises inpart from the tissue which the genetically engineered tissue-wrappedstents and tissue sheet-based vascular grafts replaces. For example,intravascular drugs are administered to the blood stream often byrepeated puncturing of the skin and veins with a needle in order toobtain a rapid systemic delivery of the drug or agent. The ability ofthe genetically engineered tissue-wrapped stents and tissue sheet-basedvascular grafts to directly deliver a therapeutic agent to the bloodstream avoids the often painful and infectious routes of administrationusing needles while at the same time maintaining a rapid systemicdelivery of the agent. For example, the genetically engineeredtissue-wrapped stents and tissue sheet-based vascular grafts comprisingstromal cells can be genetically engineered to express anticoagulationproducts to reduce the risk of thromboembolism or to expressanti-inflammatory gene products to reduce the risk of failure due toinflammatory reactions. For example, the cells of the tissue sheet canbe genetically modified to express TPA, streptokinase or urokinase toreduce the risk of clotting.

In another aspect, the cells of the tissue-wrapped stents and tissuesheet-based vascular grafts can be genetically modified to expresssoluble receptors in the form of oligomers. A number of therapeuticoligomers are currently marketed for treatment of a variety of diseasesand disorders. For example, soluble TNF-receptor oligomers sold underthe trade name ENBREL® are used to treat a number of disease anddisorders including rheumatoid arthritis, psoriatic arthritis, and thelike. ENBREL is currently delivered by injection. The invention providesthe ability to deliver ENBREL through genetically engineeredtissue-wrapped stents and tissue sheet-based vascular grafts to treat,for example, arthritis, psoriasis and the like. Products deliveredthrough genetically engineered tissue-wrapped stents and tissuesheet-based vascular grafts can be used to treat, for example: thrombusformation, inflammatory reactions, and fibrosis and calcification of thevalves.

The cells present in the tissue sheet can be engineered to express suchtherapeutic products transiently and/or under inducible control, or as achimeric fusion protein anchored to the cells of the tissue sheet. Thisexpression can be triggered by mechanical signals such as fluid shearstress or stretch, or pharmacological agents. Another example is ananchored molecule comprising the ligand binding domain of a receptor(e.g., TNF-R), wherein the anchor is provided through an intracellularand/or transmembrane domain of a receptor or receptor-like molecule,fused to the ligand binding domain of a receptor. In another embodiment,the genetically engineered tissue-wrapped stents and tissue sheet-basedvascular grafts can be used to provide a subject with a polypeptide orpolynucleotide product for which the subject is deficient. In yetanother aspect, the genetically engineered tissue-wrapped stents andtissue sheet-based vascular grafts are engineered to provide a productthat is carried by the blood; e.g., cerebredase, adenosine deaminase,and an antitrypsin.

Cells used to develop a tissue sheet can be engineered using arecombinant DNA technology to transform or transfect a host cell (i.e.,a cell that will be used to develop a tissue sheet) with a heterologouspolynucleotide, which is then clonally expanded into a robust tissuesheet. The robust tissue sheet, which expresses a product from theheterologous polynucleotide, is then implanted into a subject who isdeficient for that product. In another aspect, the tubular structuresderived from the tissue sheets of the invention can be used to delivertherapeutic agents to not only the blood, but other tissues includingother tubular tissue such as the genitourinary tract andgastrointestinal tract.

The use of the robust tissue sheet in delivery of a therapeutic productor through gene therapy has a number of advantages. First, since theculture comprises eukaryotic cells, the recombinant product will beproperly expressed and processed to form an active product. Secondly,delivery and/or gene therapy techniques are useful only if the number oftransfected cells can be substantially enhanced to be of clinical value,relevance, and utility; the robust tissue sheets of the invention allowfor expansion and amplification (via cell division) of transfected cellsinto a robust tissue sheet that is ultimately implanted into a subject.

An advantage of the genetically modified cell sheets described herein isthat the release of diagnostic and/or therapeutic agents can bedynamically controlled by triggering the cellular production by eitherpharmacological or physical means. For example, a polynucleotideencoding a product of interest (e.g., a polypeptide, antisense orribozyme molecule) can be induced to express the product under a desiredphysical condition (e.g., shear stress) that induces, for example,tissue specific regulatory elements (e.g., elastin or elastase Iregulatory elements). For example, a polynucleotide can be induced toexpress a therapeutic product as an intimal hyperplasia processes startto narrow the lumen of the stent or vascular graft due to increasingfluid shear stresses by engineering an elastin or elastase I regulatoryelement in operable association with a gene encoding a desiredtherapeutic or diagnostic product. In one aspect, the polynucleotidewould produce an anti-restenotic agent or other therapeutic agent. Inanother aspect, the polynucleotide would produce a reporter moleculethat would signal that the stent or vascular graft is loosing elasticityor viability thereby signaling that it should be replaced. Where thepolynucleotide produces an anti-restenotic agent or other therapeuticagent a self-correcting dynamic balance is achieved withoutunnecessarily eluting the therapeutic agents (which may have systemic aswell as local effects) at all times.

The tissue sheet-based constructs (e.g., the genetically engineeredvascular grafts and tissue-wrapped stents) can be implanted in vivo orused for in vitro study of biological activities in response to variousagents. Implantation in vivo of a vascular graft derived from a tissuesheet or a tissue-wrapped stent will typically be performed usingstenting and/or angioplasty procedures.

In one embodiment, the construct is delivered via catheter by piercingthrough the wall of the healthy vessel near the proximal end of adiseased area of the vessel and bypassing the diseased area by thenre-entering on the distal end of the diseased area. A percutaneousbypass would not be possible with current technologies, since normalstents do not have a membrane that would allow blood to pass throughwithout leaking (i.e., typically stents are highly porous). Moreoversynthetic grafts made from collagen or polymers or other syntheticscaffolds lack one or more of the 3 critical components of the tissueengineered stent or vessel; (1) endothelial cells; (2) adequatecompliance; and/or (3) adequate mechanical strength. In anotherembodiment, the stent is delivered on a balloon catheter and thenexpanded in place. In another, the stent or rolled vessel is insertedthrough a small atherectomy (open artery) procedure. In yet anotherembodiment, the stent or rolled vessel is delivered in a fashion similarto classic bypass, where the ends of the graft are sewn into place. Oneadvantage to this final embodiment is that sutures or other anchoringanastomotic devices can be pre-loaded onto the tissue engineeredconstruct to facilitate insertion.

The invention described herein is formed by anchoring immature tissue toa cell culture substrate to allow prolonged maturation, which increasesmechanical strength. Once the sheet is formed, it is a mechanicalbuilding block that can be used either with or without other mechanicaldevices (such as stents) in order to provide better treatment ofcardiovascular disease via catheter based intervention.

The invention provides, a tissue-coated stent assembled usingsheet-based tissue engineering techniques. In this embodiment, a robustsheet of autologous or allogenic fibroblasts is produced from cellsseeded onto a culture flask. The sheet is assembled without the need forany exogenous matrix or scaffold. The sheet is formed by adding agentsto the cell culture media that promote extracellular matrix productionand/or adhesion such that the cell layer matures into a thick sheet thatcan be manipulated. Examples of agents that promote production ofcollagen include ascorbic acid, copper, and retanoic acid. Prolongedperiods of culture increases production of extracellular matrixproteins, which provide the structural strength of the sheet. In orderto prolong culture, the sheet can be anchored to the substrate usingcoatings on the substrate such as fibrin or gelatin or by clamping thesheet with magnet clamps or control rods. The sheet can also be anchoredby growing it on a porous membrane that can later be detached anddiscarded.

The sheet is removed from the substrate and wrapped around a collapsedstent. The sheet-wrapped stent is matured for a period of time such thatthe cells of the sheet migrate onto the stent and envelope the struts ofthe stent. During this brief maturation (approximately 0–4 weeks) thestent becomes an integral part of the tissue in the sheet. During thistime, autologous endothelial cells can be seeded to the inside of thestent to make an endothelialized lumen. If the sheet is wound about thestent several revolutions, it is important to arrest the maturationprocess prior to the individual plies of the sheet fusing stronglytogether. This is important, since, when the stent is deployed in thebody, it must be expanded by a factor of 2 or 3 times its originaldiameter. Unless provisions are made such that the sheet hasextraordinary elasticity (by overexpressing elastin production in theextracellular matrix, for example), the sheet will rupture during theexpansion if it is well fused. If the sheet is only partially fused,however, the plies of the sheet will slip relative to each otherslightly, thus allowing the diameter to increase without rupture of thetissue. The sheet-wrapped stent is delivered to the diseased artery viaa catheter using techniques well known in the art. The stent is expandedusing a balloon, and the catheter is withdrawn. The stent, embedded inthe cellular sheet, is left behind. Blood clotting is reduced by thefact that the lumen of the sheet-wrapped stent is endothelialized.Migration of cells such as smooth muscle cells that cause failures byintimal hyperplasia is reduced by the presence of the sheet, which actsas a barrier. The foreign body responses to the stent are likewiseattenuated since the stent is embedded in autologous tissue. Thistechnique has been utilized in canine models and patentcy through 3months was demonstrated.

In another embodiment, the cells that form the sheet can be geneticallymodified to over-express therapeutic agents that reduce the likelihoodof failure by thrombosis or intimal hyperplasia. Similarly, the sheetscan be transfected to overexpress agents that will change thecomposition of the extracellular matrix. Elastin, for example, can beoverexpressed to increase the elasticity of the vessel and therebyreduce the likelihood of failures due to compliance mismatch at theanastomotic site.

In another embodiment, the stent can be manufactured from a resorbablematerial such that the sheet acts as a new lining for the diseased bloodvessel. In this embodiment, the stent is used only as a delivery vehicleand a temporary mechanical support. The cells in this embodiment canalso be transfected to reduce restenosis.

In another similar embodiment, the stent can be removed entirely. Sincethe tissue engineered construct is quite robust, it is possible tosupport the artery in the location of the ruptured plaque using thecylindrical roll of the cell-based sheet without any sort of stent. Inthis case, the sheet-based cylinder is formed by rolling the sheetaround a temporary steel mandrel (a temporary stent), and then allowinga prolonged maturation phase. During this maturation phase, the plies ofthe sheet are allowed to fuse more completely in order to provide a morerobust support. Since the tissue is not delivered on an expandablestent, it can be collapsed around the delivery catheter without the needfor dramatic increases in diameter (in other words, the collapsedcylinder simply unfolds as it is inflated to its normal diameter by theballoon catheter).

In another embodiment, the sheet wrapped stent, can provide a basis forpercutaneous bypass. That is, the sheet wrapped stent can be used toreroute blood flow around a blocked vessel rather than disrupting theplaque and flowing through the diseased artery. In this embodiment, thesheet-wrapped stent must be equipped with a device on the ends of thevessel that facilitates an anastomosis. This device seals the hole thatmust be made in order to make the native artery communicate with thebypass vessel. The percutaneous bypass is achieved by advancing thesheet wrapped stent to a point just proximal to the blockage, and thendirecting the delivery catheter through the wall of the diseased artery.The proximal end is then sealed using an anastomotic device as is knownin the art. The sheet wrapped stent is advanced around the diseasedartery, and is then reintroduced into the artery downstream of theblockage. Like the proximal end, the distal end must be sealed using anautomatic anastomotic device. Ordinarily, passing the catheter throughthe wall of the native artery is to be avoided at all times. What makesthis embodiment possible is that the stent is wrapped in a tissue thatmakes the bypass vessel relatively leak proof. When the native artery ispierced above and below the blockage, blood flow is re-routed around theblockage and through the wrapped stent. Similarly, the stentlessembodiment of a cylindrically formed sheet as above can be used for thispercutaneous bypass. That is, a sheet can be formed around a mandrel,matured so that the plies are well fused, and then modified such that ananastomotic device is included on each end. The entire construct isdelivered percutaneously, and blood is re-routed around the blockedartery in the vessel formed from the sheet of cells. In both of theseembodiments, the device can be further improved by genetically modifyingthe cells as described above.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

1. A tissue culture method, comprising; culturing a population ofadherent cells in a tissue culture container in the presence of one ormore tissue control rod(s), under conditions that allow the formation ofa tissue sheet comprised of living cells and extracellular matrix formedby the cells, whereby the tissue sheet is in contact with the one ormore tissue control rod(s); and removing the tissue sheet from theculture container wherein the one or more tissue control rod(s) anchorsthe tissue sheet therefore preventing unintentional detachment of thetissue sheet from the culture container.
 2. The method of claim 1,wherein the tissue sheet is removed from the culture container using theone or more tissue control rod(s).
 3. The method of claim 1, wherein theone or more tissue control rod(s) are a biocompatible material.
 4. Themethod of claim 1, wherein the one or more tissue control rod(s) aresubstantially non-porous.
 5. The method of claim 1, wherein the one ormore tissue control rod(s) are comprised of a non-adherent material. 6.The method of claim 1, wherein the one or more tissue control rod(s) arecomprised of a biodegradable material.
 7. The method of claim 6, whereinthe biodegradable material is selected from the group consisting ofcotton, polylactic acid, polyglycolic acid, a blend of polylactic andpolyglycolic acid, cat gut sutures, cellulose, gelatin, dextran, and anycombination thereof.
 8. The method of claim 1, wherein the one or moretissue control rod(s) are comprised of a non-biodegradable material. 9.The method of claim 8, wherein the non-biodegradable material isselected from the group consisting of a magnetic material, amagnetizable material, a polypropylene, a TEFLON, a steel or a steelalloy, a titanium or a titanium alloy, a polystyrene, a glass, and anycombination thereof.
 10. The method of claim 1, wherein the one or moretissue control rod(s) form a heart valve framework.
 11. The method ofclaim 1, wherein the tissue sheet has a planar shape that can be foldedor bent into a configuration by folding or bending the one or moretissue control rod(s).
 12. The method of claim 1, wherein the culturecontainer is selected from the group consisting of a tissue cultureplate, a tissue culture dish, a tissue culture flask, and a rollerbottle or other non-planar containers.
 13. The method of claim 12,wherein the culture container comprises polystyrene.
 14. The method ofclaim 12, wherein the culture container is electrostatically treated topromote adhesion of cells.
 15. The method of claim 12, wherein theculture container is treated with an agent selected from the groupconsisting of fibrin, gelatin, fibronectin, vitronectin, RDG-peptide,collagens and antibodies and any combination therof.
 16. The method ofclaim 1, wherein the population of cells is isolated from a humanbiopsy.
 17. The method of claim 1, wherein the population of adherentcells are substantially homogenous.
 18. The method of claim 1, whereinthe population of adherent cells are selected from the group consistingof fibroblasts, fibroblast precursors, endothelial cells, mesothelialcells, smooth muscle cells, mesenchymal stem cell, hematopoietic stemcells, circulating stem cells, and any combination thereof.
 19. Themethod of claim 1, further comprising substantially immobilizing the oneor more tissue control rod(s) using a magnetic field.
 20. The method ofclaim 1, further comprising using the one or more tissue control rod(s)for manipulating and/or shaping the tissue sheet into a desired tissuestructure.
 21. The method of claim 20, wherein the desired tissuestructure is selected from the group consisting of a tubular construct,a stack of tissue sheets, a heart valve and a three dimensional tissueor organ.
 22. The method of claim 1, wherein the one or more tissuecontrol rod(s) is circular.
 23. The method of claim 1, wherein the oneor more tissue control rod(s) is bent to the shape of the cell culturecontainer.
 24. The method of claim 20, wherein the one or more tissuecontrol rod(s) remains in the tissue structure.